In healthy individuals whose lungs are uninfected, lung secretions are complex non-homogeneous materials that form a viscous hydrophilic whitish gel. Mucous glycoproteins in the uninfected lung secretions contribute to the viscosity. However, in infected or purulent yellowish or greenish lung secretions as seen in cystic fibrosis, chronic bronchitis and pneumonia, both mucous glycoproteins and DNA are responsible for increasing the viscosity of the secretions. Cystic fibrosis patients who do not have concurrent bacterial or viral infections also exhibit increased mucous viscosity.
Investigators have determined that DNA, absent from healthy lung secretions, is present in large amounts from 3-14 milligrams/milliliters in purulent lung secretions. See, Chernick et al., Pediatrics, 24:739-745 (1959) and Potter et al., Am. J. Dis. Child, 100:493-495 (1960). While the viscosity of uninfected as well as infected mucous secretions in individuals with cystic fibrosis, chronic bronchitis or pneumonia was originally thought to be the result of a network of entangled glycoproteins, more recent data suggests that DNA present in the secretions contributes to the entanglement of the network. See, Shak et al., Proc. Natl. Acad. Sci., USA, 87:9188-9192 (1990). Moreover, addition of DNA to a sputum sample has been shown to increase the viscosity of DNA as described by Picot et al., Thorax, 33:235-242 (1978).
Further confirmation that DNA contributes to the high viscosity of mucous in various patient populations is based on the reduction of viscosity of lung secretions incubated in vitro with a DNA-specific degrading enzyme, partially purified bovine pancreatic DNase I. See, Armstrong et al., Lancet, ii:739-740 (1950) and Chernick et al., Pediatrics, 27:589-596 (1961). More recently, recombinant human DNase I has been used in vitro transforming a sample of nonflowing viscous sputum samples from individuals with cystic fibrosis to a flowing liquid. See, Shak et al., Proc. Natl. Acad. Sci., USA, 87:9188-9192 (1990). Aerosolized recombinant human DNase I has now been used as a short-term therapeutic treatment for individuals with cystic fibrosis and chronic bronchitis with similar efficacy to the in vitro results. See. Hubbard et al., New Engl. J. Med., 326:812-815 (1992); Aitken et al., JAMA, 267:1947-1951 (1992); and Ranasinha et al., Lancet, 342:199-202 (1993).
That enzymatic fragmentation of DNA significantly decreases the viscosity of the mucus suggests that it is not only the amount of DNA present, but its length, which contributes to the viscosity. The origin of the accumulated DNA is attributed to degenerating leukocytes, but little evidence to support this has been published. An alternative source of the DNA is by continuing renewing respiratory epithelial cells that die by programmed cell death and slough into the airway lumen. These cells may well be the source of the excessive DNA present in the mucous secretions of the airway.
The accumulation and persistence of high viscosity secretions contribute to respiratory distress and progressive lung destruction. Specifically, in diseases such as cystic fibrosis, airway secretions are a primary factor in respiratory dysfunction and ultimately contribute to the death of individuals with the disease. The secretions have been characterized as thick and highly viscous. As such, they are difficult to expectorate and contribute to reduced lung volumes and expiratory flow rates. See, for example, Welsh et al., J. Clin. Invest., 80:1523-1526 (1987). In addition, cystic fibrosis patients, as well as persons with chronic bronchitis or pneumonia, are further characterized as having chronic infections of Pseudomonas aeruginosa where despite antibiotic therapy, efficacy of treatment with aminoglycoside antibiotics is reduced. See, Potter et al., Pediatrics, 36:714-720 (1965), Vandaux et al., J. Infect. Dis., 142:586-593 (1980); and Mendelman et al., Am. Rev. Respir. Dis., 132:761-765 (1985).
The accumulation of viscous secretions in persons with cystic fibrosis is not confined to the respiratory tract, with the intestine, pancreas, biliary tract, salivary glands and genitourinary tract being similarly effected. This multisystem disorder is an autosomal recessive genetic disease due to mutation of the cystic fibrosis transmembrane conductance regulator (CFTR) on the long arm of chromosome 7. The gene occupies 250 kilobases with 27 exons, and encodes a single polypeptide of 1480 amino acids which is N-glycosylated. The CFTR is predominantly expressed in epithelial cells but mRNA can also be detected at much lower levels in leukocytes, skeletal muscle, as well as fetal liver, kidney, heart, and brain. See, Hubbard et al., New Eng. J. Med., 326:812-815 (1992).
The CFTR is a transmembrane anion channel for both chloride and bicarbonate ions that opens in response to cAMP, but has also been shown to regulate chloride conductance through other channels. See, Riordan et al., Science, 245:1066 (1989). Over 200 specific mutations of the CFTR gene have been reported, most of which result in the phenotype of cystic fibrosis (CF) in the homozygous state and are asymptomatic when heterozygous. The most common mutation is a three base pair deletion at position 508 (delF508).
In airways of cystic fibrosis individuals, the mutation results in the dysfunction of the CFTR ion channel so that secretion of both bicarbonate and chloride ions are markedly reduced. This inability of epithelia to respond with chloride secretion results in the above-described sequelae. The cellular consequence of the retention of chloride and bicarbonate in the epithelial cells is alkalinization of the cytosol and defective acidification of organelles such as the endoplasmic reticulum, where pH-dependent glycosylation is altered. See, Barash et al., Nature, 352:70-73 (1991) and Elgavish et al., Am. J. Physiol., 263:176-186 (1992). Thus, the epithelial cells have a baseline pH that is higher than in comparable normal cells.
Intracellular acidification has been correlated with endonuclease activation in a cell line designated HL-60, a promyelocytic leukemia line, following exposure and damage by the calcium ionophore ionomycin as described by Barry et al., Biochem. Biophys. Res. Commun., 186:782-789 (1992). More recently, the same authors have observed digestion of DNA in HL-60 cells in vitro following exposure to etoposide, an inhibitor of topoisomerase II (Barry et al., Cancer Res., 53:2349-2357 (1993). Within 3.5 hours after exposure to etoposide, the cells underwent concentration-dependent intracellular acidification where the intracellular pH reached maximal acidification of 0.15 pH units. As a result, in a portion of the treated cell population, DNA was shown to be digested through activation of DNase II that requires an acidic environment for activation. Acidification of the cells correlated with the time course of appearance of DNA digestion. The authors suggested that intracellular pH homeostasis may play a role in digestion of genomic DNA that is characteristic of a pathway of cell death referred to as apoptosis or programmed cell death. Investigators using other in vitro systems have also correlated acidification with DNA degradation. See, Caceres-Cortes et al., J. Biol. Chem., 269:12084-12091 (1994) and Li et al., J. Biol. Chem., 270:3203-3211 (1995).
In a recent paper, an inhibitor of protein isoprenylation, lovastatin, was shown to induce DNA degradation in HL-60 cells by first causing a dose-dependent decrease in pH. See, Perez-Sala et al., J. Biol. Chem., 270:6235-6242 (1995). This biological response was observed in a subset of cells whole pH was 0.9 pH units below control levels. In contrast, activation of the sodium/hydrogen antiporter pump resulted in an increase in pH which was sufficient to prevent or arrest DNA degradation. However, the authors stated that lovastatin-induced intracellular acidification was not due to the complete inhibition of the pump. Thus, other biological mechanisms are probably functioning simultaneously in HL-60 cells to promote DNA degradation.
Control of intracellular pH is accomplished through a variety of ion channels and pumps, including the sodium/hydrogen exchanger, the vacuolar proton ATPase, and CFTR in selected cell populations. In one intracellular pH-effecting therapeutic regimen with cystic fibrosis patients having a mutant CFTR, treatment with aerosolized amiloride, an inhibitor of the sodium/hydrogen exchange pump, resulted in an decrease of sputum viscosity with a concomitant improvement in elasticity. See, Knowles, et al., New Eng. J. Med., 322:1189-1194 (1990).
Exposure of a sarcoma cell line to weak acids was shown to result in intracellular acidification only if the extracellular pH was kept at pH 6.5 or less but not if cells were maintained at physiological pH. See Karuri et al., Brit. J. Cancer, 68:1080-1087 (1993). With low extracellular pH, the cells were not viable. This effect was probably due to the property that at extracellular physiological pH, the cells' ion-compensating mechanisms were active, while at a lower pH, the cells failed to compensate. Moreover, the authors did not see any acidification in tumors in vivo following intraperitoneal injection of a solution of the weak acids.
Despite the observations described above, the cellular mechanism or mechanisms responsible for the secretion of undegraded genomic DNA found in the sputum of cystic fibrosis patients and the presence of a mutated CFTR protein was equivocal. As a result, the pathophysiological connection between abnormal chloride secretion and the clinical manifestations of cystic fibrosis were previously unclear.
The methods of the present invention are based on the discovery that the cytoplasmic alkalinization caused by the mutant CFTR interferes with the process of DNA fragmentation that depends upon acidification. The methods rely on the findings that acidification is not only a concomitant of or correlated with, but an absolute prerequisite for degradation of DNA ultimately leading to cell death. In addition, an in vitro model system of the present invention has confirmed the direct relationship between acidification and DNA degradation. As such, the failure to degrade DNA in an intracellular alkaline environment contributes to the pathogenesis of cystic fibrosis as senescent CFTR-mutant epithelial cells and/or leukocytes lyse and release undegraded, viscous DNA into the airway lumen.
Fragmentation of DNA has been characterized as one step in a process of programmed cell death, also referred to as apoptosis. Apoptosis was first described by Wyllie et al., Int. Rev. Cytol., 68:251-306 (1980) and is now recognized to be a tightly-regulated physiological process. Although the specific signals regulating apoptosis are poorly understood, cell death by apoptosis is characterized by preservation of the cell membrane (preventing spillage of pro-inflammatory cell contents), crosslinking of cellular proteins by a transglutaminase to form a cornified envelope, DNA fragmentation, condensation and fragmentation of the nucleus into small apoptotic bodies, and expression of markers targeting the cells/apoptotic bodies for phagocytosis (phosphatidylserine, ICAMs). This process is quite efficient, as exemplified by the clearance of enormous numbers of inflammatory cells during the resolution of streptococcal pneumonia.
However, in cystic fibrosis, chronic bronchitis and pneumonia, these processes break down resulting in the clinical profiles described above. While the symptoms of cystic fibrosis can be attributed to the mutant CFTR protein causing an increase in intracellular pH, the accumulation of viscous DNA in individuals suffering from chronic bronchitis and pneumonia is probably due to chronic metabolic alkalosis which results in an increase in intracellular pH, thereby inhibiting the DNA degradation pathway that is facilitated by an acidic intracellular environment. Therefore, in these patients, senescent cells will not undergo apoptosis-induced DNA fragmentation but rather will lyse releasing the high molecular weight DNA into the lumen of the airways.
The novel methods of this invention for ameliorating viscous mucous-associated diseases including cystic fibrosis, chronic bronchitis and pneumonia are based on an in vitro model cell culture system. The model system comprises epithelial cells transfected with the normal and mutant CFTR gene, the latter of which is referred to as delF508. Treatment with exogenously applied acidifying agents have now been discovered to convert a mutant CFTR-transfected cell line, maintained at physiological pH in which apoptosis-induced DNA fragmentation is inhibited, to a cell that exhibits a decrease in intracellular pH resulting in DNA fragmentation.
Thus, the methods of this invention provide a new therapeutic regimen for individuals having a viscous mucous-associated disease by inducing DNA fragmentation and consequently programmed cell death in cells that would have previously undergone lysis without concomitant DNA degradation.